title: Experimental identification of microRNA-140 targets by silencing and overexpressing miR-140.
abstract: MicroRNAs (miRNAs) are short noncoding RNA molecules regulating the expression of mRNAs. Target identification of miRNAs is computationally difficult due to the relatively low homology between miRNAs and their targets. We present here an experimental approach to target identification where the cartilage-specific miR-140 was overexpressed and silenced in cells it is normally expressed in separate experiments. Expression of mRNAs was profiled in both experiments and the intersection of mRNAs repressed by miR-140 overexpression and derepressed by silencing of miR-140 was identified. The intersection contained only 49 genes, although both treatments affected the accumulation of hundreds of mRNAs. These 49 genes showed a very strong enrichment for the miR-140 seed sequence implying that the approach is efficient and specific. Twenty-one of these 49 genes were predicted to be direct targets based on the presence of the seed sequence. Interestingly, none of these were predicted by the published target prediction methods we used. One of the potential target mRNAs, Cxcl12, was experimentally validated by Northern blot analysis and a luciferase reporter assay.
authors.affiliation: School of Biological Sciences, University of East Anglia, Norwich, NR4 7TJ, United Kingdom.
authors.forename: Francisco Esteban
authors.initials: FE
authors.lastname: Nicolas
authors.forename: Helio
authors.initials: H
authors.lastname: Pais
authors.forename: Frank
authors.initials: F
authors.lastname: Schwach
authors.forename: Morten
authors.initials: M
authors.lastname: Lindow
authors.forename: Sakari
authors.initials: S
authors.lastname: Kauppinen
authors.forename: Vincent
authors.initials: V
authors.lastname: Moulton
authors.forename: Tamas
authors.initials: T
authors.lastname: Dalmay
mesh_terms: D000818:Animals; D002460:Cell Line; D054377:Chemokine CXCL12 / Q000235:genetics; D002645:Chickens; D005347:Fibroblasts; D051379:Mice; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*
chemical_list: D054377:Chemokine CXCL12; D035683:MicroRNAs